The overall objective of this proposed research is to investigate mammalian vitamin B6 metabolism and its regulation. The in vivo uptake and rate of metabolism of radioactive pyridoxine by a number of mouse tissues will be investigated. Particular emphasis will be placed on those tissues that show most rapid vitamin B6 uptake, metabolism, and release of metabolites into plasma for transport to other tissues. The regulation of the availability of the coenzyme forms of B6 will be investigated at the level of their enzymatic synthesis and degradation and of their transport. The binding of pyridoxal and pyridoxal-P to specific proteins in tissues and in plasma, and the role of these binding proteins in vitamin B6 transport will be investigated. The plasma binding protein will be identified, purified, and characterized as to its affinity for the vitamin B6 forms and its inhibition by various compounds. Three enzymes involved in vitamin B6 metabolism will be purified. These are pyridoxal (pyridoxine) kinase from mammalian liver and pyridoxine-P oxidase and vitamin B6-P phosphatase from human erythrocytes. The kinetic properties of these enzymes and their inhibition or activation by various compounds will be investigated. The possibility of product inhibition, feedback inhibition, and substrate activation of these enzymes will be studied to elucidate their role in control of vitamin B6 metabolism. Vitamin B6 metabolism is known to be altered by some drugs, such as ethanol or its metabolites. The effect of these drugs on the binding of the vitamin B6 forms to proteins and on the activity of the purified enzymes will be investigated.